Use of binding partners for 5-ht5 receptors for the treatment of neurodegenerative and neuropsychiatric disorders

ABSTRACT

The present invention relates to the use of binding partners for 5-HT5 receptors for the treatment of neuropathological, in particular neurodegenerative and/or neuropsychiatric, disorders, which can occur, in particular, in cerebral ischemia, stroke, epilepsy and seizures in general, chronic schizophrenia, other psychotic disorders, dementia, in particular Alzheimer&#39;s dementia, demyelinizing disorders, in particular multiple sclerosis, and brain tumors. The invention also relates to processes for the identification and characterization of such binding partners, in particular in the form of screening processes.

The present invention relates to the use of binding partners for 5-HT5receptors for the treatment of neuropathological disorders andassociated indications, symptoms and dysfunctions and to processes forthe identification and characterization of binding partners of thistype.

At least seven different receptor classes mediate the manifoldphysiological activities which are ascribed to an involvement of theneurotransmitter serotonin (5-hydroxytryptamine, abbreviated 5-HT).According to an internationally recognized classification, they aredesignated by 5-HT1, 5-HT2,5-HT3,5-HT4,5-HT5,5-HT6 and 5-HT7. Most ofthese classes moreover include receptor types which can bedifferentiated further. Thus the 5-HT1 class includes receptors whichcan be divided into at least five subclasses, which are designated by5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess F. G. and Martin I. L.,Neuropharmacology 33:275-317 (1994)).

The 5-HT5 class was described for the first time by Plassat et al., TheEMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992). 5-HT5A and 5-HT5Breceptors are differentiated (Erlander et al., Proc. Natl. Acad. Sci.USA 90:3452-3456 (1993)). Only small sequence homologies exist between5-HT5 and other 5-HT receptors. The pharmacological profile of thesereceptors differs markedly. Using molecular biology techniques, thelocalization of 5-HT5 receptors was possible in the olfactory bulb, inthe hippocampus, in the cortex, in the cerebral ventricles, in thecorpus callosum and in the cerebellum. By means of immunohistochemicalmethods, it was possible to show that 5-HT5 receptors are principallyexpressed on astrocytes (Carson et al., GLIA 17:317-326 (1996)).Astrocytes are directly adjacent to the basal membrane of braincapillaries of the blood-brain barrier. An abnormal astrocyteendothelium structure accompanies a loss of the blood-brain barrier. Theexact significance of the astrocytes is unclear. They appear to lookafter transport tasks and connective functions. Reactive astrocytes wereobserved in connection with reactive gliosis in a number of pathologicalbrain changes and neuropsychiatric disorders. As a result of braininjuries, they change their morphologies. The protein expression patternchanges and growth factors are produced. In vitro investigations oncultured astrocytes have allowed the detection of 5-HT5receptor-mediated responses. It is thus to be suspected on the one handthat they are involved in recovery processes of the brain afterdisorders, but on the other hand it is also not to be excluded that theycontribute to the creation of damage or even to an increase in damage.

CNS disorders nowadays concern large sections of the population. Inparticular on account of the increase in elderly people, the numbers ofpatients are increasing continuously. Neuropathological conditions suchas cerebral ischemia, stroke, epilepsy and attacks in general, chronicschizophrenia, other psychotic disorders, dementia, in particularAlzheimer's dementia, demyelinizing disorders, in particular multiplesclerosis, and brain tumors lead to damage to the brain and the neuronaldeficits associated therewith.

Therapeutic treatments of the neurodegenerative and neuropsychiatricdisorders outlined were up to now directed at various membrane receptorswith the aim of compensating deficits in neurotransmission processes.Indeed, it was possible to achieve neuroprotective effects withserotonogic compounds in animal models of neuropathological conditions,such as ischemia, cerebral stroke and excitotoxicity. In some cases, itwas also possible to observe favorable effects on emotionaldisturbances, such as depression or anxiety states. Mention may be madehere, for example, of 5-HT1A agonists, such as buspirone or the compound8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), which ischaracterized as a selective 5-HT1A receptor ligand. These activecompounds, however, only decrease neurological deficits to a limitedextent. There is still no effective therapy at present.

It is therefore an object of the present invention to make possible thetreatment of neuropathological disorders with adequate efficacy andminor side effects.

Surprisingly, it has now been found that treatment of the above diseaseconditions and associated indications, symptoms and dysfunctions is madepossible by specific use of substances having binding affinities for5-HT5 receptors.

One subject of the present invention is therefore the use of bindingpartners for 5-HT5 receptors for the preparation of an agent for thetreatment of neuropathological disorders and associated indications,symptoms and dysfunctions.

Neuropathological disorders are understood according to the invention asmeaning disorders which are accompanied by neurological deficits, i.e. acondition characterized by neurological deficiency symptoms. The term“disorder” in the sense according to the invention designates anomalieswhich, as a rule, are regarded as pathological conditions and can revealthemselves in the form of certain signs, symptoms and/or dysfunctions.The treatment according to the invention can be directed at individualdisorders, viz. anomalies or pathological conditions, but a number ofanomalies which are causally connected to one another can be combined togive patterns, i.e. syndromes, which can be treated according to theinvention.

This condition can exist temporarily, progressively or persistently.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a plot of bound [³H]-5-CT as a function of the [³H]-5-CTconcentration.

FIG. 2 shows 5-HT and 5-CT induced an increase in the [³⁵S] GTPγSbinding to cell membranes over basal values.

FIG. 3 a shows a plot of the percentage of total binding as a functionof the GDP concentration.

FIG. 3 b shows the [³⁵S] GTPγS binding stimulation.

FIG. 4 shows the [³⁵S] GTPγS binding stimulation for the percentage ofthe maximal activity as a function of the 5-HT concentration.

According to the invention, the treatment of neurodegenerative and/orneuropsychiatric disorders is preferred. These disorders occur, inparticular, in neuropathological syndromes, as a rule syndromes causedby brain damage, for example cerebral ischemia, stroke, epilepsy andseizures in general, chronic schizophrenia, other psychotic disorders,dementia, in particular Alzheimer dementia, demyelinizing disorders, inparticular multiple sclerosis, and brain tumors. The invention inparticular also relates to the use of 5-HT5 binding partners for thetreatment of those forms of the abovementioned disorders in whoseformation and/or course 5-HT5 receptors are involved, i.e. disorderswhich are modulated by a 5-HT5 receptor activity.

According to a further aspect of the present invention,neuropathological disorders are treated which accompany a glialreaction. The use according to the invention relates in particular tothe modulation of a glial reaction.

An advantageous action of the binding partners is seen in the preventiveor acute treatment of neurological deficits, which are observed inpatients who suffer from psychiatric disorders, such as epilepsy,psychosis, e.g. psychoses of the acute exogenous reaction type orconcomitant psychoses of organic or exogenous cause, e.g. after trauma,especially brain lesions and diffuse brain damage, in metabolicdisorders, infections, and endocrinopathies; endogenous psychoses, suchas schizophrenia, and schizotypic and delusional disorders; effectivedisorders, such as depression, mania and manic depressive conditions;and mixed forms of the psychoses described above; senile dementia andsenile dementia of the Alzheimer type, and in the treatment orprevention of demyelinization processes.

The binding partners according to the invention are efficacious, inparticular with respect to the treatment of ischemic damage, e.g. as aresult of brain and spinal cord trauma and vascular occlusion or heartfailure.

Especially to be mentioned here is stroke (synonym: cerebral apoplexy,cerebral or apoplectic insult, cerebral stroke). Transitory ischemicattacks, reversible ischemic neurological deficits, prolonged reversibleischemic neurological deficits, partially reversible ischemicneurological symptoms and also persistent complete cerebral infarcts canbe treated according to the invention. The treatment of acute forms isparticularly advantageous according to the invention.

One or more of the changes in nerve tissues listed below underlie theforms of neuropathological disorders which can be preferably treatedaccording to the invention: degeneration or death of neurons, inparticular of the ganglial cells, e.g. tigrolysis, indistinctness of thenuclear membrane, plasmolysis, cytoplasm vacuolization and encrustation,parenchymal necroses of the brain, cerebral edema, changes to neuronscaused by oxygen deficiency, atrophy, morphological changes, such asdemyelinization, in particular medullary sheath disintegration,perivascular infiltrates, glial proliferation and/or glial scarring;degeneration of the Substantia nigra.

The indication to be treated according to the invention is oftencharacterized by a progressive development, i.e. the conditionsdescribed above change in the course of time, as a rule the degree ofseverity increases and conditions further to already existing conditionscan occur.

By means of the treatment according to the invention ofneuropathological disorders or of the conditions underlying them, anumber of further signs, symptoms and/or dysfunctions which areconnected with the neuropathological disorders can be treated, i.e. inparticular accompany the disorder conditions described above. Theseinclude, for example, shock lung, brain nerve losses, e.g. retrobulbarneuritis, eye muscle paralysis, syllabication, spastic paralysis,cerebella symptoms, sensitivity, bladder and rectal disorders, euphoria,dementia, hyper- and akinesia, absence synchynesis, small-step gait,bent posture of trunk and limbs, pro-, retro- and lateropulsion, tremor,lack of facial expression, monotonous speech, depression, apathy, labileor rigid affectivity, impaired spontaneity and resoluteness, slowedthinking, poor association ability; muscular atrophy.

A treatment in the sense according to the invention comprises not onlythe treatment of acute or chronic signs, symptoms and/or dysfunctionsbut also a preventive treatment (prophylaxis), in particular as arelapse or phase prophylaxis. The treatment can be achievedsymptomatically, for example as symptom suppression. It can be carriedout short-term, be carried our medium-term, or it can also be along-term treatment, for example in the context of a maintenancetherapy.

The term “binding partner for 5-HT5 receptors” describes substanceswhich bind to 5-HT5 receptors and can therefore also be designated as5-HT5 receptor ligands.

Binding is understood as meaning any molecular interaction between thebinding partner and the receptor, in particular under physiologicalconditions. As a rule, these are conventional interactions, whichinclude electrostatic attraction, hydrogen bonding, hydrophobic bonds,van-der-Waals forces or metal complex-like coordinative bonds. Inaddition to the above-mentioned, reversible molecular interactions,irreversible interactions between binding partner and receptor can alsobe possible, such as, for example, covalent bonds.

According to one embodiment, binding partners which can be usedaccording to the invention competitively inhibit the binding ofcomparison binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT(5-carboxamidotryptamine), to 5-HT5 receptors.

Competitive inhibition is understood as meaning that binding partnerswhich can be used according to the invention compete with a comparisonbinding partner, in the present case, for example, 5-HT or 5-CT, forbinding to the receptor, i.e. the binding of one prevents the binding ofthe other.

According to a further embodiment, binding partners which can be usedaccording to the invention inhibit the binding of comparison bindingpartners, such as 5-HT (5-hydroxytryptamine) or 5-CT(5-carboxamidotryptamine) to 5-HT5 receptors noncompetitively.

Noncompetitive inhibition is understood as meaning that binding partnerswhich can be used according to the invention modulate, via their bindingto the receptor, the binding of a comparison binding partner, in thepresent case, for example, 5-HT or 5-CT, in particular lower its bindingaffinity.

At least in the case of competitive inhibition, i.e. of reversiblebinding, the principle applies that the displacement of one bindingpartner by another increases with decreasing binding affinity of the oneor increasing binding affinity of the other with respect to thereceptor. More expediently, therefore, binding partners which can beused according to the invention have a high binding affinity for 5-HT5receptors. A binding affinity of this type allows, on the one hand, aneffective displacement of naturally occurring binding partners for 5-HT5receptors, such as, for example, serotonin (5-hydroxytryptamine, 5-HT)itself, where the necessary concentration of binding partner which canbe used according to the invention for the binding of a certain amountof this binding partner to 5-HT5 receptors decreases with increasingbinding affinity. With respect to medical use, binding partners aretherefore preferred whose binding affinity is so great that these can beadministered in justifiable amounts in the course of an effectivemedical treatment as an active compound. Binding partners which can beused according to the invention are therefore preferably administered indaily doses of approximately 0.01 to 100 mg/kg of body weight and inparticular of approximately 0.1 to 15 mg/kg of body weight on parenteraladministration and 1 to 30 mg/kg of body weight on oral administration.

The competition experiments referred to above, with which thatconcentration of binding partner which can be used according to theinvention is determined in vitro which displaces 50% of anothercomparison binding partner from the receptor binding site (IC₅₀ values),offer one possibility of expressing the binding affinity. Thus thecompetitive inhibition of the binding of 5-CT to 5-HT5 receptors canalso be evaluated to the effect that binding partners which canpreferably be used according to the invention have half-maximalinhibition constants IC₅₀ of less than 10⁻⁵ M, preferably of less than10⁻⁶ M and in particular of less than 10⁻⁷ M.

The binding affinity of binding partners which can be used according tothe invention can also be expressed by means of the inhibition constantK_(i), which is in general likewise determined in vitro usingcompetition experiments. For the binding of 5-HT5 receptors, bindingpartners which can be used according to the invention preferably haveK_(i) values of less than 10⁻⁶ M, advantageously of less than 10⁻⁷ M andparticularly preferably of less than 10⁻⁸ M. K_(i) values of compoundswhich can be used according to the invention lie, for example, in therange from 1·10⁻⁷ M to 7·10⁻⁷ M or in the range from 1·10⁻⁸ M to 1·10⁻⁷M.

Binding partners which can be used can bind with a lower, an essentiallyidentical, or a higher affinity to 5-HT5 than to a specific receptorwhich is different from 5-HT5.

Thus binding partners for 5-HT5 receptors with respect to the useaccording to the invention in particular include those whose bindingaffinity for 5-HT5 receptors compared with the affinity for 5-HT1receptors, in particular 5-HT1A, 5-HT1B and/or 5-HT1D, is so high thatthey are advantageously suitable for the use according to the invention.This does not necessarily presuppose a comparatively more selectivebinding to 5-HT5 receptors, even though selective binding partners for5-HT5 receptors are a particular embodiment of the present invention.

For example, binding partners can be used which have high affinity bothfor 5-HT5 and for 5-HT1 receptors, in particular for 5-HT1A, 5-HT1Band/or 5-HT1D. In this connection, high affinity means K_(i) values as arule in the range from 1·10⁻⁹ M to 1·10⁻⁶ M. According to a particularembodiment, binding partners which can be used in the high affinityrange have a binding profile for 5-HT receptors which is characterizedby a binding affinity to 5-HT5 which, in comparison to other bindingaffinities of this range is essentially identical or only slightly less.Factors of 10 or less can be advantageous.

Selective binding partners which can be used according to the inventionhave binding affinities for 5-HT5 receptors which are larger than forone or more 5-HT receptors which are different from 5-HT5, i.e. inparticular receptors allocated to the abovementioned 5-HT receptorclasses 5-HT1,5-HT2,5-HT3,5-HT4,5-HT6 and 5-HT7. If the binding affinityfor 5-HT5 receptors of a binding partner is greater than that of a 5-HTreceptor which is different from 5-HT5, we speak of a selective bindingof these binding partners to 5-HT5 receptors in relationship to the 5-HTreceptor which is different from 5-HT5. Particular binding partners arethose whose binding affinity for 5-HT5 receptors is greater than for atleast one and in particular all 5-HT1 receptors, in particular for5-HT1A, 5-HT1D and/or 5-HT1B receptors. Binding partners whose bindingaffinity for 5-HT5 receptors is greater than for all 5-HT receptorswhich are different from 5-HT5 constitute a further particular class ofbinding partners according to the invention.

Selectivity is understood as meaning the property of a binding partnerto bind preferably to 5-HT5 receptors.

It is decisive for the selectivity outlined above that the bindingaffinities for 5-HT5 receptors on the one hand and for one or more of5-HT receptors which are different from 5-HT5 on the other hand areadequately different. Affinity differences are preferred according towhich binding affinity ratios of at least 2, advantageously of at least5, particularly advantageously of at least 10, preferably of at least20, particularly preferably of at least 50 and in particular of at least100 are present.

According to a further embodiment, binding partners which can be usedaccording to the invention bind selectively to 5-HT5 receptors havingthe advantageous binding affinities described above in relation to oneor more 5-HT receptors other than 5-HT5.

According to a further embodiment, binding partners which can be usedaccording to the invention bind selectively to 5-HT5 receptors havingthe advantageous binding affinities described above in relation to all5-HT receptors other than 5-HT5.

Binding partners are particularly advantageous which bind to 5-HT5receptors which are expressed by glia cells and in particular byastrocytes with the affinities and selectivities described above.

According to the invention, the human receptor variant is a preferredtarget for the binding partners employed according to the invention.

The binding of binding partners according to the invention to 5-HT5receptors is coupled to an effector function. Binding partners can actagonistically or antagonistically and partly agonistically and/or partlyantagonistically.

Agonists are designated as compounds according to the invention whichcompletely or partially imitate the activity of 5-HT on 5-HT5 receptors.

Antagonists are designated as compounds according to the invention whichcan block the agonistic activity of 5-HT on 5-HT5 receptors.

According to a preferred embodiment of the present invention, bindingpartners are employed whose binding at least to 5-HT5 receptors ofh5-HT5-transfected CHO cells brings about a change in theagonist-induced stimulation of GTP binding to membrane-bound G proteins,a change in intracellular calcium levels, a change in agonist-inducedinduction of phospholipase C activity and/or a change in cAMPproduction. As far as the change in intracellular calcium levels isconcerned, the use of binding partners which bring about an increase inintracellular calcium levels represents a particular embodiment of theinvention.

This embodiment also includes binding partners which are active in knownanimal models for neurodegenerative and neuropsychiatric processes.

Preferred binding partners are those which are also selective for 5-HT5receptors in relation to their effector function in the sense describedabove.

Compounds which can be used according to the invention are described,for example, in DE 197 24 979.5. These are 3-substituted3,4,5,6,7,8-hexahydropyrido[3′,4′:4,5]thieno-[2,3-d]pyrimidinederivatives of the formula I

in whichR¹ is a hydrogen atom, a C₁-C₄-alkyl group, an acetyl group, aphenylalkyl C₁-C₄ radical, where the aromatic is optionally substitutedby halogen, C₁-C₄-alkyl, trifluoromethyl, hydroxyl, C₁-C₄-alkoxy, amino,cyano or nitro groups or is a phenylalkanone radical, where the phenylgroup can be substituted by halogen,R² is a phenyl, pyridyl, pyrimidinyl or pyrazinyl group, which isoptionally mono- or disubstituted by halogen atoms, C₁-C₄-alkyl,trifluoromethyl, trifluoromethoxy, hydroxyl, C₁-C₄-alkoxy, amino,monomethylamino, dimethylamino, cyano or nitro groups, and which canoptionally be fused to a benzene nucleus, which can optionally be mono-or disubstituted by halogen atoms, C₁-C₄-alkyl, hydroxyl,trifluoromethyl, C₁-C₄-alkoxy, amino, cyano or nitro groups and canoptionally contain 1 nitrogen atom, or to a 5- or 6-membered ring whichcan contain 1-2 oxygen atoms,A is NH or an oxygen atom,

Y is CH₂, CH₂—CH₂, CH₂—CH₂—CH₂ or CH₂—CH,

Z is a nitrogen atom, carbon atom or CH, where the bond between Y and Zcan also be a double bond,and n is the number 2, 3 or 4and their salts with physiologically tolerable acids.

Further compounds which can be used according to the invention aredescribed, for example, in DE 196 36 769.7. These are 3-substituted3,4,5,6,7,8-hexahydropyrido[4′,3′:4,5) thieno-[2,3-d]pyrimidinederivatives of the formula I

in whichR¹ is a hydrogen atom, a C₁-C₄-alkyl group, an acetyl or benzoyl group,a phenyl C₁-C₄-alkyl radical, where the aromatic is optionallysubstituted by halogen, C₁-C₄-alkyl, trifluoromethyl, hydroxyl,C₁-C₄-alkoxy, amino, cyano or nitro groups, or is a naphthyl-C₁-C₃-alkylradical, a phenyl-C₂-C₃-alkanone radical or a phenylcarbamoyl-C₂-alkylradical, where the phenyl group can be substituted by halogen,R² is a phenyl, pyridyl, pyrimidinyl or pyrazinyl group, which isoptionally mono-, di- or trisubstituted by halogen atoms, C₁-C₄-alkyl,trifluoromethyl, trifluoromethoxy, hydroxyl, C₁-C₄-alkoxy, amino,monomethylamino, dimethylamino, cyano or nitro groups, and which canoptionally be fused to a benzene nucleus which can optionally be mono-or disubstituted by halogen atoms, C₁-C₄-alkyl, hydroxyl,trifluoromethyl, C₁-C₄-alkoxy, amino, cyano or nitro groups and canoptionally contain 1 nitrogen atom, or to a 5- or 6-membered ring whichcan contain 1-2 oxygen atoms,or can be substituted by a phenyl-C₁-C₂-alkyl or -alkoxy group, wherethe phenyl radical can be substituted by halogen, or a methyl,trifluoromethyl or methoxy group,A is NH or an oxygen atom,B is hydrogen or methyl,C is hydrogen, methyl or hydroxyl,X is a nitrogen atom,

Y is CH₂, CH₂—CH₂, CH₂—CH₂—CH₂ or CH₂—CH,

Z is a nitrogen atom, carbon atom or CH, where the bond, between Y and Zcan also be a double bond,and n is the number 2, 3 or 4,and their salts with physiologically tolerable acids.

5-HT5-specific antibodies can also be utilizable as 5-HT5 bindingpartners. They can be polyclonal antisera, monoclonal antibodies,antibody fragments, such as F(ab), Fc, etc., chimeric and recombinantantibodies. Such antibodies can be prepared in a manner known per se. Asan immunogen, 5-HT5 receptor can be used as such or antigenic fragmentsthereof, as a rule coupled to customary carrier proteins.

Further low molecular weight 5-HT5 binding partners, usually syntheticcompounds, can be used advantageously in many respects.

Aptamers, i.e. nucleic acids, as a rule oligonucleotides, havingsufficient affinity for 5-HT5 receptors, can also be used as bindingpartners.

The use according to the invention is not restricted to theabovementioned binding partners. Rather, any substance which binds to5-HT5 receptors in the manner described above, can be used according tothe invention as a 5-HT5 binding partner.

Assays for the determination of binding affinities of test substancesfor 5-HT5 receptors are known in principle. This can be carried out, forexample, by assessing the competitive inhibition of the binding of acomparison binding partner to 5-HT5 receptors by the substance to beinvestigated. Suitable comparison binding partners are known ligands for5-HT receptors, such as 5-HT or 5-CT or LSD. These are expedientlylabeled such that their binding to 5-HT receptors can be monitoredanalytically using standard methods. Radioactive and optical markers arepreferred. In binding studies on 5-HT5 receptors, according to theinvention, 5-CT or LSD, in particular in the form of [³H]-LSD, is used.The binding affinities can be expressed as half-maximal inhibitionconstants IC₅₀ or as inhibition constants K_(i). This process ispreferably used for primary screening. SPA technology or FlashPlatetechnology is preferably used.

The binding to binding partners to be investigated can also bedetermined directly on 5-HT receptors. The inhibition constants K_(i)expressing binding affinity can be determined, for example,calorimetrically, i.e. by measurement of the binding energy released.

Effector functions can also be assessed qualitatively or quantitativelyboth in vitro and in vivo with the aid of known functional assays.

The assessment of an agonistic activity can be based on all thoseeffects which are produced by the binding of 5-HT to 5-HT5 receptors. Itis preferred according to the invention to assess the effects on thebinding of GTP to G proteins, on intercellular calcium levels, on thephospholipase C activity and/or on the cAMP production. These processesare preferably used for secondary screening. Here too, SPA or FlashPlatetechnology is advantageously used.

GTP binding to G proteins can be investigated by using a nonhydrolyzableanalog of GTP, for example [³⁵S] GTPγS, whose binding can beinvestigated radiologically. This investigation is preferably carriedout on membranes having 5-HT5 receptors.

For the measurement of intracellular calcium levels, it is possible toemploy suitable calcium probes, as a rule calcium chelating agents, forexample fluorescing compounds, such as Fura 2-acetylmethyl ester orfluo-3-AM. This investigation is preferably carried out on cell cultureshaving 5-HT5 receptors, in particular on individual cells.

The phospholipase C activity can be determined by means of the reactionscatalyzed by it, for example, the incorporation of myoinositol, whichfor detection purposes is preferably radiolabeled as [³H)-myoinositol,or the conversion of PPIP₂ to IP₃, where the PPIP₂ is also preferablyradiolabeled as [³²P)PIP₂]. These investigations are preferably carriedout on individual cells having 5-HT5 receptors.

cAMP production can be determined with the aid of the cAMP bindingprotein. This investigation is preferably carried out on individualcells having 5-HT5 receptors.

If appropriate, the effector function is also determined, i.e. theactivity of binding partners according to the invention for other 5-HTreceptors. This expediently takes place taking into account the bindingaffinities determined for 5-HT5 and other 5-HT receptors, i.e. inparticular taking into account the selectivity.

The present invention therefore also relates to processes for theidentification and characterization of binding partners which can beused according to the invention. These and further processes which aresimilarly suitable can form the basis for in vitro screening processeswith which it is possible from a large number of different compounds topick out those which, with respect to future use, appear to be the mostpromising. For example, by means of combinatorial chemistry, extensivesubstance banks can be prepared which comprise myriads of potentialactive compounds. The inspection of combinatorial substance librariesfor substances having desired activity is automatable. Screening robotsare used for the efficient evaluation of the individual assays, whichare preferably arranged on microtiter plates. Thus the present inventionalso relates to screening processes, i.e. both primary and secondaryscreening processes, in which preferably at least one of the processesdescribed below is used. If a number of processes are used, this can beshifted in terms of time or simultaneously carried out on one and thesame sample or on different samples of a substance to be investigated.

A particularly effective technology for carrying out processes of thistype is the scintillation proximity assay, called SPA for short, knownin the field of active compound screening. Kits and components forcarrying out this assay can be obtained commercially, for example fromAmersham Pharmacia Biotech. In principle, solubilized or membrane-boundreceptors are immobilized on small fluoromicrospheres containingscintillation substance. If, for example, a radioligand binds to theimmobilized receptors, the scintillation substance is stimulated to emitlight, since the spatial vicinity between scintillation substance andradioligand is specified.

A further particularly effective technology for carrying out processesof this type is the FlashPlate® technology known in the field of activecompound screening. Kits and components for carrying out this assay canbe obtained commercially, for example from NEN® Life Science Products.This principle is likewise based on microtiter plates (96-well or384-well), which are coated with scintillation substance.

The abovementioned assays are known in principle to the person skilledin the art.

A first process according to the invention is used for the determinationof the affinity and/or selectivity of binding partners for 5-HT5receptors. For this purpose, the binding partner is brought into contactwith 5-HT5 receptors and the binding affinity is determined.

For the determination of selectivities, the binding affinity of thebinding partner to be investigated to other 5-HT receptors is determinedin the same manner—if appropriate using the ligands specific for therespective receptor—and the values obtained are compared.

A further process according to the invention relates to thedetermination of the activity of binding partners for 5-HT5 receptors,i.e. the determination of agonistic, partly agonistic, antagonisticand/or partly antagonistic action. For this purpose, the binding partneris brought into contact with 5-HT5 receptors and the effects caused bythe binding are assessed.

According to a preferred embodiment, binding partners are subjected to aprimary screening by determining their binding affinity to 5-HT5receptors using the [³H]-5-CT or [³H]-LSD competition experimentdescribed above. Those binding partners which have an inhibitionconstant IC₅₀ in the range of 10⁻⁶M or less are then subjected to asecondary screening by assessing their effector function in the mannerdescribed above, in particular with respect to GTP binding and/or theintracellular calcium levels. Finally, the binding partners selected inthis way can be subjected to a counter-screening for selectivitydetermination by determining their binding affinity to further 5-HTreceptors essentially in the manner described above—but optionally usingthe ligands specific to the respective receptors. For example,[³H]-8-hydroxydipropylaminotetralin ([³H]-8-DPAT) can be used forbinding studies to 5-HT1A receptors, while 5-HT1B and 5-HT1D receptorscan be investigated using [³H]-5-CT.

5-HT5 receptors are preferably made available in the form of cellularsystems, i.e. in the form of membranes, cells, cell colonies, tissues ororgans which carry 5-HT5 receptors. Cellular systems of this type canexpress 5-HT5 receptors by nature, but they can also be induced toexpress 5-HT5 by suitable genetic manipulation, e.g. by transfection. Inthe context of the preferred embodiment of the present inventionconcerning h5-HT5, it is possible to use for this purpose, inparticular, the coding sequence described in Rees S. et al, FEBS Letters335:242-246 (1994) (accession number X81411). Human glioma cell linesare preferred as natural cellular systems having 5-HT5 receptors. Of theh5-HT5-transfected heterologous cell lines, those are preferred whichexpress the h5-HT5 gene. Mention may be made, for example, ofh5-HT5-transfected CHO cells, h5-HT5-transfected human kidney cells, inparticular h5-HT5-transfected HEK293 cells, or h5-HT5-transfected C-6glioma cells.

For the determination of selectivity, affinity and activity of bindingpartners according to the invention, it is also possible to use braintissue sections and native membranes from brain parts.

If radio labels are employed, the assessment of tissue sections ispreferably carried out autoradiographically.

The neuroprotective action is preferably determined on animal models ofneurodegenerative and neuropsychiatric processes.

Preferred animal models are those for cerebral stroke and cerebraldisorders in the case of multiple infarct, for example cerebralischemias as a result of an occlusion of the carotid artery or themiddle cerebral artery (MCA occlusion), fore brain ischemias and hypoxiatolerance tests, anxiolytic models, for example active compound-inducedconvulsions, electroshock- or isolation-induced aggression, models ofantiepileptic activity, for example electroshock-, active compound- ornoise-induced attacks and genetic models, excitotoxic neurodegenerationmodels and demyelinization models.

In the context of the treatment, the use according to the invention of5-HT5 binding partners comprises a process. In this process, theindividual to be treated, preferably a mammal, in particular a human,agricultural or domestic animal, is administered an efficacious amountof one or more 5-HT5 binding partners, as a rule corresponding topharmaceutical and veterinary medical practice. Whether such a treatmentis indicated and in what form it has to take place depends on theindividual case and is subject to medical assessment (diagnosis), thesigns, symptoms and/or dysfunctions present, risks of developing certainsigns, symptoms and/or dysfunctions, and additionally includes furtherfactors.

As rule, the treatment is carried out by single or repeated dailyadministration, if appropriate together or in alternation with otheractive compounds or active compound-containing preparations, such thatan individual to be treated is administered a daily dose ofapproximately 0.001 g to 10 g, preferably of approximately 0.001 g toapproximately 1 g.

The invention also relates to the preparation of pharmaceuticalcompositions for the treatment of an individual, preferably a mammal, inparticular a human, agricultural or domestic animal. The bindingpartners according to the invention are usually administered in the formof pharmaceutical compositions which comprise a pharmaceuticallytolerable excipient with at least one inhibitor according to theinvention and, if appropriate, further active compounds. Thesecompositions can be administered, for example, by the oral, rectal,transdermal, subcutaneous, intravenous, intramuscular or intranasalroute.

Examples of suitable pharmaceutical formulations are solidpharmaceutical forms, such as powders, granules, tablets, pastilles,sachets, cachets, coated tablets, capsules such as hard and soft gelatincapsules, suppositories or vaginal pharmaceutical forms, semisolidpharmaceutical forms, such as ointments, creams, hydrogels, pastes orpatches, and also liquid pharmaceutical forms, such as solutions,emulsions, in particular oil-in-water emulsions, suspensions, forexample lotions, injection and infusion preparations, eye and ear drops.Implanted delivery devices can also be used for the administration ofbinding partners according to the invention. In addition, liposomes,microspheres or polymer matrices can also be used.

In the production of the compositions, binding partners according to theinvention are usually mixed or diluted with an excipient. Excipients canbe solid, semisolid or liquid materials which are used as a vehicle ormedium for the active compound.

Suitable excipients include, for example, lactose, dextrose, sucrose,sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. Inaddition, the formulations can comprise pharmaceutically acceptablevehicles or customary excipients, such as lubricants, for exampletallow, magnesium stearate and mineral oil; wetting agents; emulsifyingand suspending agents; preservatives, such as methyl and propylhydroxybenzoates; antioxidants; antiirritants; chelating agents;pan-coating auxiliaries; emulsion stabilizers; film-forming agents;gel-forming agents; flavor-masking agents; flavor corrigents; resins;hydrocolloids; solvents; solubilizers; neutralizing agents; permeationaccelerators; pigments; quaternary ammonium compounds; refatting andsuper fatting agents; ointment, cream or oil bases; siliconederivatives; spreading auxiliaries; stabilizers; sterilizing agents;suppository bases; tablet excipients, such as binders, fillers,lubricants, disintegrants or coatings; propellants; drying agents;opacifying agents; thickeners; waxes; plasticizers; white oils. Arelevant embodiment is based on expert knowledge, such as is presented,for example, in Fiedler, H. P., Lexikon der Hilfsstoffe für Pharmazie,Kosmetik and angrenzende Gebiete (Encyclopedia of excipients forpharmacy, cosmetics and related areas], 4th Edition, Aulendorf:ECV-Editio-Kantor-Verlag, 1996.

The present invention is illustrated in greater detail by means of thefollowing examples, without being restricted thereto.

REFERENCE EXAMPLE 1 h5-HT5 Receptor-Expressing HEK293 Cells and CHOCells

The gene coding for the human 5-HT5 receptor was isolated from humantissues in a known manner by means of 3′-5′-RT-PCR (RACE system,Boehringer Mannheim). The gene sequence was then inserted into a plasmidcarrying the neomycin resistance gene (pcDNA3; Invitrogen, Germany) andamplified in E. coli according to the manufacturer's instructions. Apreparation of the resulting plasmid was mixed with Lipofectamin® (GibcoLife-Sciences, Germany), and HEK293 cells were incubated with a thinlayer of this transfection mixture in petri dishes (2.5 cm). Thetransfection mixture was then replaced by neomycin-containing culturemedium. Surviving cells were further cultured in DMEM-F12 medium whichwas supplemented with 10% fetal calf serum, 2 mM glutamine andantibiotics (90 mg of streptomycin, 90 mg of penicillin). The cells weregrown to confluence under 5% CO₂, 95% atmospheric humidity and 37° C.

h5-HT5 receptor-expressing CHO cells are obtained analogously.

REFERENCE EXAMPLE 2 Cell Membrane Preparation

The method used here essentially follows known methods for thepreparation of cell membranes from cells (Findlay J. B. C. and Evans W.H. Biological Membranes, Practical Approach (1987)). The cells culturedaccording to Reference Example I were carefully scraped off the surfaceof the culture vessel and centrifuged in DMEM-F12 medium at 180×g for 10min. The cell pellets obtained were resuspended in 5 mM tris HCl buffercontaining 5 mM EDTA, 5 mM EDTA, 0.1 mM PMSF and 3 mM benzamidine (pH:7.6; buffer A) and incubated at 4° C. for 15 min. The cell suspensionwas homogenized (6×3s) in an Ultraturrax® (15,000 rpm) and centrifugedat 1000×g and 4° C. for 1 min. The pellet was resuspended in buffer Aand, as described above, homogenized and centrifuged. The supernatantsfrom both steps were collected and centrifuged at 40,000×g and 4° C. for20 min. The pellet was resuspended and homogenized in buffer A (1×15s).The membrane suspension was centrifuged at 40,000×g and 4° C. for 20min. The resulting pellet was resuspended in buffer A containing 10%glycerol and 1% bovine serum albumin. Aliquots were frozen and stored at−80° C. until use.

REFERENCE EXAMPLE 3 Kinetics of the Saturation Binding of [³H]-5-CT

The methodology is essentially known (Ress S. et al., FEBS Letters335:242-246 (1994). Membranes obtained according to Reference Example 2(200 μl) were incubated in a total volume of 600 μl in 100 mM tri5-HClcontaining 1 mM EDTA (pH: 7.7; buffer B) with increasing concentrationsof [³H]-5-CT (96 Ci/mmol), 10 μm methiothepine being added for thedetermination of the specific binding, while methiothepine was not addedfor the determination of the total binding. The mixture was incubated at30° C. for 90 min. The samples were then filtered, a Skatron® filtrationsystem and GF/B filters embedded in 0.3% polyethyleneimide being used.The filters were washed at 4° C. with 9 ml of buffer B. Theradioactivity retained on the filters was measured by means of liquidscintillation counting, 5 ml of Ultima-Gold (packard) being used.

REFERENCE EXAMPLE 4 a) [³H]-5-CT Binding Competition

The experiments on binding competition were carried out essentiallyfollowing known investigations (Rees et al., 1994). Membranes obtainedaccording to Example 2 (200 μl) were incubated in the presence of 2 nM[³H)-5-CT in a total volume of 600 μl in buffer B with increasingconcentrations of selected compounds. After an incubation time of 75 minat 30° C., the samples were filtered at 4° C. through GF/B filtersembedded in 0.3% polyethyleneimide using buffer B. The filters werewashed with 9 ml of buffer B. The radioactivity retained on the filterswas determined as in Reference Example 3. The total binding was definedas that binding of the radioligand which was observed without additionof further compounds. This nonspecific binding was defined as thatbinding of [³H]-5-CT which was observed in the presence of 10 μMmethiothepine. Similar systems can also be used which, as a result ofuse of microtiter plates, allow a high sample throughput and secondaryscreening.

The saturation parameters of the [³H]-5-CT binding was determined bothby nonlinear regression analysis and from linear plots using theSigmaPlot software (Jandel Scientific, Germany). Competition curves wereset up in which the radioactive binding is expressed as a percentageproportion of the total binding. Half maximal inhibition constants IC₅₀and Hill-coefficients (n_(H)) were determined by means of nonlinearregression analysis.

b) Identification of H5-HT5 Receptor Ligands by HTS Using FlashPlateTechnology

96-well FlashPlates which are coated with h5-HT5 membranes can beobtained from Bio Signal Inc. (Canada). [3H)-LSD was diluted to asuitable concentration in Tris HCl buffer which contains 10 mM MgCl₂,0.5 mM EDTA and 0.5% of BSA. The radioligand solution was added to thewells (25 ml), which either contained or did not contain test compound.The plates were incubated at room temperature for 180 minutes and theradioactive signal was measured using a micro β-counter (Wallac). Thenonspecific binding was determined using methiothepine. [3H]-LSD has anaffinity of 12 nM. With increasing binding affinity of the testcompound, the radioactive signal of [3H]-LSD decreased.

REFERENCE EXAMPLE 5 Determination of the Agonist-Induced Stimulation of[³⁵S)GTPγS Binding

[³⁵S]GTPγS binding assays are known. The present assay was carried outfollowing the previously described method of Hilf, G. and Jakobs, K. H.(Eur. J. Mol. Pharmacol. 225:245-252 (1992)). Active compound-inducedchanges in the [³⁵S]GTPγS binding to membranes of HEK293 cells stablytransfected with the h5-HT5 receptor gene were measured (see ReferenceExamples 1 and 2). The cell membranes (12 μg) were incubated with 50 nMtriethanolamine HCl buffer (pH: 7.5) containing 6.75 mM MgCl₂, 150 nMNaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP and [³⁵S] GTPγS. Following a60-minute incubation at 30° C. with or without addition of the activecompounds to be tested, the test mixture (100 μl) was rapidly filteredthrough GF-B filters using a Skatron® filtration device. The filterswere rapidly washed with 50 mM tris HCl buffer (9 ml; pH: 7.5; 4° C.)containing 100 mM NaCl and 5 mM MgCl₂. The radioactivity retained on thefilters was determined by means of scintillation spectrometry, UltimaGold scintillation fluid being used. Similar systems which allow a highthroughput and secondary screening as a result of use of microtiterplates can likewise be used.

The active compound activities were expressed as a percentage proportionof the basic binding measured in the absence of the active compound. Thematching of the curves was carried out using software for nonlinearregression analysis (SigmaPlot, Jandel Scientific, Germany) according tothe general equation E=(L×E_(max))/(L+EC₅₀), in which E is the action, Lis the ligand concentration, E_(max) is the maximum action and EC₅₀ isthat concentration which induces 50% of the maximal action.

REFERENCE EXAMPLE 6 Determination of Agonist-Induced Changes ofIntracellular Calcium Levels

The method is known (Kao J. P. Y. Methods in Cell Biology 40:155-181(1994)). As described in Reference Example 1, h5-HT5 receptor-expressingHEK293 cells were grown in culture vessels. The cells were carefullyscraped off before they were confluent. The cells were labeled with Fura2 by incubating at room temperature with Fura 2-acetylmethyl ester(Sigma). The cells were centrifuged at 180×g for 10 min and resuspendedin DMEM-FI2 medium without serum and incubated at 37° C., 5% CO₂ and 95%atmospheric humidity for 45 min.

Intracellular calcium levels were determined with a fluorescencemicroscope which was equipped with a suitable filter exchange system(Olympus/Hamamatsu). The fluorescence ratio (340 nm/380 nm) wasdetermined using the Argus® software. The intracellular calcium levelswere observed for a short time in individual cells without the additionof active compounds and then 30 min after addition of the activecompound to be tested. Similar systems which permitted a high throughputand secondary screening as a result of the use of microtiter platescould likewise be used.

The modulation of intracellular Ca²⁺ levels can be assessed analogouslyin HTS. For this, h5-HT5 receptor-expressing CHO cells were culturedovernight in 96-well plates (30,0000 [sic]-80,000 cells/well). The cellswere labeled for one hour using HEPES buffer containing 1 mM Fluo-3-AM,10% pluronic acid and 2.5 mM probencid, and washed. A test compound wasadded to each well. For the determination of the calcium levels, thefluorescence intensity was read off using a fluorometrically operatingplate reader (Fluorometric Imaging Plate Reader; FLIPR).

REFERENCE EXAMPLE 7 Determination of the Agonist-Induced Phospholipase CActivity

The method is essentially known (Garcia-Ladona F. J. et al., Neuroreport4:691-694 (1993)). The cells were incubated with 0.125 μM[³H]myoinositol for 24 h. Unincorporated [³H]myoinositol was removedfrom the medium and replaced by Krebs-Henseleit buffer containing 10 mMLiCl. After incubation for 10 minutes, the active compound to be testedwas added. After 45 min, the reaction was stopped by replacing thestimulation medium by distilled water. If tissue samples are used, asimilar procedure is employed (Garcia-Ladona et al., 1993). The cellswere frozen and stored at −80° C. The production of [³H]inositolmonophosphate was determined by means of known chromatographic methods.A similar method can be used with tissue miniprisms. The determinationof the phospholipase C stimulation was likewise carried out in a similarmanner by preparing membrane fractions, as described in ReferenceExample 2, and incubating with [³²P]PIP₂ and active compounds. In thiscase, the production of IP3 was determined. Known processes were alsooptimized in order to use systems based on microtiter plates.Commercially obtainable materials allow extension to analyses with ahigh throughput and the carrying-out of secondary screening.

REFERENCE EXAMPLE 8 Determination of the Agonist-Induced Change in cAMPProduction

The method used is essentially known (Strada S. S. et al., Methods inNeurotransmission receptor analysis: 89-110 (1990)). Cells wereincubated in culture medium without serum and antibiotics for 10 min.The medium was heated at 95° C. for 15 min in order to stop thereaction. The cell samples were frozen and stored at −80° C. cAMP levelswere determined using commercially obtainable kits which use the cAMPbinding protein. Known processes were also optimized in order to usesystems based on microtiter plates. Commercially obtainable materialsallow extension to analyses with a high throughput and the carrying-outof secondary screening.

REFERENCE EXAMPLE 9 Tissue Preparation

90 min after administration of the active compound (orally,intraperitoneally, intravenously or intracerebroventricularly), theexperimental animals were decapitated. The entire brain was rapidlyremoved from the skull, frozen on dry ice and stored at −80° C. Ratbrain sections (15 μm) were obtained in a cryostat at −20° C., appliedto gelatin-coated slides and stored at −30° C. until use.

REFERENCE EXAMPLE 10 Neuroprotective Action MCA Occlusion

The neuroprotective activity of the test compounds was investigated forexperimentally caused cerebral stroke in a standard model. Theexperiments were performed on male Long Evans rats. Under nitrousoxide-assisted halothane anesthesia, the middle cerebral artery (MCA)was severed and permanently ligated distally, as described in theliterature. The resulting infarct volume was determined 22 hours afterMCA occlusion.

REFERENCE EXAMPLE 11 Neuroprotective Action Experimental Brain Trauma ofthe Rat

The lateral fluid percussion method (McIntosh T K, Neuroscience 28, 233244, 1989) was used in order to produce an experimental brain trauma inthe rat which is suitable for the testing of potential neuroprotectivesubstances. By means of this technique, controlled and consistent tissuedamage was produced underneath the site of the impact, which includedthe neocortex, the hippocampus and the thalamus.

The right parietotemporal region of the skull was exposed in theanesthetized rat. The skull was trepanned through the parietal cortex(about 4 mm diameter), the dura being left intact. A Luer lockattachment was fixed to the skull using dental cement through thetrepanation. After the dental cement had hardened, the rat was connectedto the fluid percussion apparatus according to McIntosh. This consistedof a cylinder filled with 0.9% NaCl solution, one end of the cylinderbeing closed by a piston, while the other end was connected via apressure transducer to a Luer lock attachment. Using this, thecounterpart on the rat was tightly closed so that a liquid column wasformed adjacent to the dura. A metal pendulum was allowed to impact ontothe piston from a predetermined height so that a brief blow of thecompressed liquid column on the surface of the rat brain occurred. Highpressures of about 2.5-2.9 bar were used.

The rats were sacrificed 14 days after traumatization for removal of thebrain. After fixation of the brains, sections of thickness 30 mm wereprepared in a freezing microtome and stained using Toluidine Blue. Forthe quantification of the neuronal damage, cell counts were carried outbilaterally in the rostral hippocampus; the proportion of intact cellsin the dentate gyrus was determined on the traumatized (right) and the(left) opposite side. The quotient of the cell count in the ipsilateralhippocampus and the number in the contralateral hippocampus wasindicated.

REFERENCE EXAMPLE 12 Synthesis of Tested Binding Partners a)3,4,5,6,7,8-Hexahydro-7-methyl-3-[2-(4-(3-trifluoromethylphenyl)piperazin-1-yl)ethyl]pyrido[4′,3′:4,5]thieno[2,3-d]-pyrimidin-4-one×2HCl(compound A)

The compound was prepared by heating2-ethoxymethylenamino-3-cyano-6-methyl-4,5,6,7-tetrahydrothieno-[2,3-c]pyridineor2-ethoxymethylenamino-3-carboethoxy-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridineunder reflux with 1-(2-aminoethyl)-4-(3-trifluoromethylphenyl)piperazine in an inert solvent such as ethanol. The reaction mixture wasworked up, and the product having a melting point of 309-312° C. wasdeposited by precipitating with ethereal hydrochloric acid.

A further possibility for the preparation consisted in first heating theabovementioned starting compounds with ethanolamine under reflux in aninert solvent such as ethanol. After the reaction mixture had beenworked up, the3,4,5,6,7,8-hexahydro-3-(2-hydroxy)ethyl-7-methylpyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-oneobtained was reacted, for example, with thionyl chloride in order tointroduce a suitable leaving group, so that the reaction of theresulting3,4,5,6,7,8-hexahydro-3-(2-chloro)-ethyl-7-methylpyrido-[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-onewith N-(3-trifluoromethylphenyl)piperazine in an inert solvent such asxylene under basic conditions (e.g. potassium carbonate) afforded thedesired product after working up the reaction mixture.

b)3,4,5,6,7,8-Hexahydro-6-ethyl-3-[2-(4-(1-naphthyl)piperazin-1-yl)ethyl]pyrido[3′,4′:4,5]thieno[2,3-d]pyrimidin-4-one×3HCl×2H₂O(compound B)

Preparation was carried out in principle as described for compound A.

2-Ethoxymethylenamino-3-carboethoxy-5-ethyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridinewas heated under reflux with 1-(2-aminoethyl)-4-(1-naphthyl)piperazinein an inert solvent such as ethanol. The reaction mixture was worked up,and the product converted into the hydrochloride was isolated with amelting point of 298-300° C.

Alternatively,3-(2-chloroethyl)-6-ethyl-3,4,5,6,7,8-hexahydropyrido[3′,4′:4,5]thieno[2,3-d]pyrimidin-4-onewas reacted under basic conditions with N-(1-naphthyl)piperazine in aninert solvent such as xylene.

c)3,4,5,6,7,8-Hexahydro-7-methyl-3-[2-(4-(7-methoxynaphth-1-yl)piperazin-1-yl)ethyl]pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-2-one×2HCl×H₂O(compound C)

Preparation can be carried out analogously to compound A.

Compounds A, B and C and also the intermediates needed for theirpreparation are known or can be synthesized from analogous startingmaterials according to the preparation methods described in theliterature (F. Sauer and P. Stanetty, Monatsh. Chem. (1975), 106(5),1111-1116; K. Gewald et al., Chem. Ber. 99, 94-100 (1966), PatentApplications DE 196 36 769.7 and DE 197 24 979.5).

EXAMPLE 1

According to Reference Example 3, the binding affinity of [³H]-5-CT to5-HT5 receptors was determined. FIG. 1 shows a plot of bound (³H]-5-CTas a function of the [³H]-5-CT concentration. A dissociation constant ofK_(d)=0.570 nM was determined. Depending on the clonal cell line, thereceptor binding density (B) varied in a range from 900-28,000 fmol/mgof protein.

EXAMPLE 2

According to Reference Example 4, the binding affinities ofserotoninergic compounds were determined by means of [³H]-5-CT bindingcompetition. By means of the IC₅₀ values obtained, the inhibitionconstants K_(i) of the following compounds were determined(K_(i)=IC₅₀/(1+C/K_(d))), where C is the concentration of [³H]-5-CT andK_(d) was determined according to Example 1):

Compound K_(i) [M] R(+)-8-OH-DPAT 1.25 · 10−⁷ 5-CT 1.44 · 10−⁹ CompoundA 6.61 · 10−⁷ Compound B 1.24 · 10−⁷ Compound C 2.37 · 10−⁷

EXAMPLE 3

According to Reference Example 5, the active compound-induced binding ofGTP to G proteins was investigated. The coupling of 5-HT5 receptors to Gproteins in HEK293 cells was evident. The typical serotoninergicagonists 5-HT and 5-CT induced an increase in the [³⁵S]GTPγS binding tothe cell membranes of over 40% above the basic value (see FIG. 2). The5-HT5 receptor needs GDP for the coupling to G proteins, which ismediated by agonists (see FIG. 3A). The 5-HT effect was dose-dependent(see FIG. 4) with an EC₅₀ of 2.6 μM.

EXAMPLE 4

According to Reference Example 10, the neuroprotective action ofselected 5-HT5 binding partners was tested. The active compounds wereadministered intravenously, first as a bolus and then as a maintenanceinfusion, 90 minutes after MCA occlusion. The following results wereachieved:

Infarct volume, % of the control [mean Dose, i.v. value ± SD, (n)] P,Active [mg/kg + Active Student's t compound mg/kg/h] Placebo compoundtest, bilateral R(+)-8-OH-D 2 + 1 100 ± 11 (12) 72 ± 32 (12) 0.0086 PATCompound A 4 + 2 100 ± 25 (12) 62 ± 18 (10) 0.0013 Compound B 2 + 1 100± 20 (12) 66 ± 19 (9) 0.0015 Compound C 1 + 0.5 100 67 <0.05

EXAMPLE 5

According to Reference Example 11, an experimental brain trauma of therat was induced using the lateral fluid percussion method. Theprotective action of the active compounds tested on the survival ofhippocampal neurons is expressed as the quotient of the neuron count ofthe trauma side to the neuron count of the contralateral side; the mean(m) and the mean error of the mean (S_(m)) is in each case indicated

P, Dose Quotient m ± S_(m) (n) Student's Active [mg/ Active t test,compound kg i.p.] Placebo compound bilateral Compound 20 + 20 0.33 ±0.05 (10) 0.56 ± 0.05 (10) <0.05 C [15 + 120 min post] Compound 20 + 200.44 ± 0.03 (17) 0.64 ± 0.03 (12) <0.05 B [15 + 120 min post]

1. The use of at least one binding partner for 5-HT5 receptors for thepreparation of an agent for the treatment of neuropathological disordersand associated indications, symptoms and dysfunctions.
 2. The use asclaimed in claim 1 in cerebral ischemia, stroke, epilepsy and attacks ingeneral, chronic schizophrenia, other psychotic disorders, dementia, inparticular Alzheimer's dementia, demyelinizing disorders, in particularmultiple sclerosis, and brain tumors.
 3. The use as claimed in claim 1or 2, wherein the K_(i) values of the binding partner for its binding to5-HT5 receptors are less than 10-⁶ M, preferably less than 10-⁷ M and inparticular less than 10-⁸ M.
 4. The use as claimed in one of claims 1 to3, wherein the binding affinity of the binding partner for 5-HT5receptors is greater than for one or more 5-HT receptors other than5-HT5.
 5. The use as claimed in one of the preceding claims, wherein thebinding affinity of the binding partner for 5-HT5 receptors is greaterthan for 5-HT1A, 5-HT1D and/or 5-HT1B receptors.
 6. The use as claimedin one of the preceding claims, wherein the binding partnercompetitively inhibits the binding of 5-CT to 5-HT5 receptors.
 7. Aprocess for the determination of the affinity of binding partners for5-HT5 receptors, where the binding partner is brought into contact withcell systems having 5-HT5 receptors and the binding affinity isdetermined.
 8. A process for the determination of the activity ofbinding partners for 5-HT5 receptors, where the binding partner isbrought into contact with cell systems having 5-HT5 receptors and atleast one binding partner-induced action is determined.
 9. A process asclaimed in claim 8, where the binding of GTP to G proteins,intracellular calcium levels, the phospholipase C activity and/or thecAMP production are determined.
 10. A process as claimed in either claim8 or 9, wherein human glioma cell lines or h5-HT5-transfectedheterologous cell lines are used.
 11. A process as claimed in claim 10,wherein h5-HT5-transfected CHO cells, h5-HT5-transfected human kidneycells, or h5-HT5-transfected C-6 glioma cells are used.
 12. An in vitroscreening process for the identification of a 5-HT5 receptor bindingpartner, where at least one process as claimed in claims 7 to 11 isused.
 13. A process as claimed in claim 12 for the identification ofbinding partners as defined in claims 1 to 6.